T-pa mutant gk1l

ABSTRACT

The invention concerns a recombinant DNA which codes for a protein with the domains G, K1 and L of t-PA in which the sequences coding for the domains K2 and F of the wild-type t-PA gene or the sequences derived therefrom within the scope of the degeneration of the genetic code are completely deleted according to the exact exon/intron borders on the t-PA gene. The invention also relates to a process for the production of a recombinant DNA according to the present invention. In addition the invention concerns vectors containing this recombinant DNA as well as cells which are transformed with vectors according to the present invention or with the DNA according to the present invention. Furthermore the invention provides a protein with fibrinolytic properties by expression of a DNA sequence according to the present invention in suitable host cells which consists of the amino acid sequences of the domains G, K1 and L of t-PA in this order and which, if desired, is glycosylated as well as a process for its production. Finally the invention also concerns a fibrinolytic agent containing a protein according to the present invention.

Human tissue plasminogen activator (t-PA) is a serine protease with amolecular weight of 68000 daltons which converts the pro-enzymeplasminogen into the active serine protease plasmin. Plasmin dissolvesfibrin which is the main component of the protein matrix of coagulatedblood. t-PA has a high affinity for fibrin and is also activated byfibrin (see Fibrinolysis 2 (1988), 133-142). t-PA is therefore of greatmedical interest.

An advantage of t-PA compared to other known plasminogen activators,such as e.g. urokinase or streptokinase, is the ability to stimulate itscatalytic activity by fibrin (see J. Biol. Chem. 257 (1982), 2912-2919;Biochem. Biophys. Acta 755 (1983) 531-533).

t-PA (amino acid sequence cf. Vehar et al., Bio/Technology 2 (1984)1051-1057) in its single-chain form consists of a heavy chain (H chain)and a light chain (L chain) which are held together by a disulphidebridge. The two-chain form is formed from a single-chain precursor formby specific cleavage with plasmin or other proteases between the aminoacids (aa) 275 and 276. The heavy L chain of 32000 daltons weightcontains the enzymatically active region which has homologies to otherserine proteases such as urokinase or plasmin (Proc. Natl. Acad. Sci.USA 81 (1984), 5335-5339). The domains on the H chain of 39000 daltonsweight are the finger domain (F) with homology to fibronectin (aa 1-49),the growth factor domain (G) with homology to mouse and human epidermalgrowth factor (aa 50-87) and two kringle domains, K1 (aa 88-175) and K2(aa 176-262) with homology to the kringle structures in plasminogen.

Concerning the function of the individual domains of the H chain it isalready known that only the domains K2 or/and F but not the domain K1are responsible for binding of t-PA to fibrin and thus for the abilityto stimulate the catalytic activity of t-PA in the presence of fibrin(EP-A-0 234 051).

It is known from EMBO 7 (1988) 2731-2740 that the activity of a t-PAmutant containing the complete domains K1 and F as well as a part ofdomain K2 of the H chain can also be stimulated by fibrin.

It is thus not clear to what extent the individual domains of the Hchain effect the activity of the t-PA molecule with regard to fibrin andto what extent they cause a stimulation of the plasminogen cleavingactivity.

The object of the present invention is to provide a t-PA mutant whichhas greater plasminogen cleaving activity than t-PA and whose catalyticactivity can also be stimulated by fibrin or fibrinogen.

This object is achieved according to the present invention by theproduction of a recombinant DNA which codes for a protein with thedomains GK1L of t-PA whereby the sequences coding for the domains K2 andF or sequences derived therefrom within the scope of the degeneration ofthe genetic code are completely deleted i.e. according to the exactexon/intron borders on the t-PA gene. The nucleotides 715 to 972 and 199to 339 of the t-PA cDNA are therefore missing from the recombinant DNAaccording to the present invention (numbering according to Nature 301,(1983), 214-221). Surprisingly the ability to stimulate the catalyticactivity by fibrin is preserved in GKIL although in the t-PA mutant GK1Lthe domains K2 and F of the H chain which are regarded as absolutelyessential for stimulating the activity are completely missing from thegene product of the recombinant DNA according to the present invention.Surprisingly the catalytic activity of a supernatant from cells whichexpress GK1L is even significantly higher than the activity of asupernatant from cells which express t-PA.

The invention also provides a process for the production of recombinantDNA according to the present invention from a DNA sequence coding fort-PA or for a t-PA mutant which contains more than the domains G, K1 andL by deletion of those sequences which do not code for the domains G, K1and L while maintaining the exact exon/intron borders on the t-PA gene.A process is particularly preferred in which the deletion of the DNAcoding for the domains K2 and F is carried out by site-directedmutagenesis.

The invention also provides a recombinant vector which contains one orseveral copies of the recombinant DNA according to the presentinvention. In this connection a preferred embodiment is a vector whichis suitable for the expression of the recombinant DNA in eukaryoticcells. A particularly preferred embodiment of the invention is aeukaryotic vector with the GK1L gene which contains the early SV40promoter and a mouse dhfr⁻ gene. However, the plasmid pSV-GKIL accordingto the present invention is most preferred.

Furthermore the invention provides a cell line which is transformed withthe recombinant DNA according to the present invention or a vectoraccording to the present invention. Particularly preferred is aeukaryotic cell line, most preferred being a CHO dhfr⁻ cell line (e.g.ECACC 88072103), which contains a recombinant DNA according to thepresent invention or a vector according to the present invention.

The invention also provides a protein with fibrinolytic properties whichconsists of the amino acid sequences of the domains G, K1 and L of t-PAin this order and which is glycosylated, if desired. The invention alsoincludes a process for the production of a protein with fibrinolyticproperties in which a recombinant DNA according to the present inventionor a vector according to the present invention is expressed in suitablehost cells and the expression product is obtained from the culturemedium or by lysis of the host cells. In this connection a process ispreferred in which the protein according to the present invention isobtained from eukaryotic host cells, preferably CHO dhfr⁻ cells, in aglycosylated form. Surprisingly a supernatant from CHO dhfr⁻ cells,which are transformed with the plasmid pSV-GKIL according to the presentinvention and which secrete GK1L, has a higher catalytic activity thanthe supernatant from cells which are transformed with a correspondingexpression vector pSV-FGK1K2L on which the wild-type t-PA gene ispresent.

A process is particularly preferred in which host cells are used whichare cultured in a medium containing aprotinin.

Surprisingly in this case the activity as well as the extent ofstimulation by fibrin is higher in the supernatant of host cells whenthe culture medium contains aprotinin.

Finally the invention provides a fibrinolytic agent which contains aprotein according to the present invention.

The following Examples in conjunction with the FIGS. 1 and 2 areintended to elucidate the invention further.

They show:

FIG. 1 the production of plasmid pSV-GKlL,

FIG. 2 a comparison of the fibrinogen-stimulated catalytic activity ofwild-type t-PA and GK1.

FIG. 3 shows the structure and amino acid sequence of wild-type t-PAmolecule.

EXAMPLE 1 Production of a t-PA derivative in which the finger andkringle 2 domains are deleted (GK1)

The production of the deletion mutant FGK1L from t-PA cDNA was carriedout by deletion of the kringle 2 domain by site-directed mutagenesisaccording to the process from Bio/Technology 2 (1984), 636- 639. pePA133 (produced according to EP-A 0242836) served as the starting plasmidon which the t-PA nucleotide sequence 190-1809 is present. Themutagenesis primer 1 (5' GCCTGCTCTGAGTCCACCTGCGGC 3') was used to removethe nucleotides 715 to 972 (exons VIII and IX) (according to thenumbering of t-PA cDNA in Nature 301 (1983), 214-221). A plasmid p7745which codes for the deletion mutant FGK1L was then isolated by colonyhybridization with the mutagenesis primer 1 and sequenced. Areconstitution of the t-PA signal sequence was necessary for theexpression later in eukaryotic cells.

At first the FGK1L cDNA was provided with the leader sequence and the3'UT (3' untranslated region) corresponding to the original cDNAsequence. For this plasmid p7.1, DSM 4719, which contained in thepolylinker of pUC12 the 5'UT (5' untranslated region) up to position 77,the leader sequence and the N-terminal sequence of t-PA up to andincluding nucleotide position 208, is cleaved with Pst I and Hind III(ca. 2.7 kb). In addition fragments containing the following t-PA cDNAsequences were isolated: a Pst I/Hae II fragment from p7745 whichcomprises nucleotide positions 209 to 421 as well as a Hae II/EcoRIfragment with the nucleotide positions 421 to 1273 in which nucleotidepositions 715 to 972 are deleted by mutagenesis and an Eco RI/Hind IIIfragment with nucleotide positions 1274-2165 from pePA 98.1 (productionsee EP-A 0 242 836).

The fragments were ligated and transformed in E. coli coli DSM 3689.Plasmids carrying transformants were selected in the culture medium byaddition of 50 μg/ml ampicillin. The correct plasmid, denoted pePA 159,was verified by restriction enzyme analysis. The FGK1L cDNA could beisolated from this plasmid as an XbaI-HindIII fragment (with a signalsequence but without its own polyadenylation site). This fragmentcontains a 7 nucleotide 5' untranslated region (5' UT) and the 3'untranslated region (3' UT) up to the BglII site at position 2160(Nature 301 (1983) 214-221). The mutagenesis primer 2 (5'GATCTTACCAATGCAGCGAGC3') was used for the deletion of the finger domainF (exon IV) from FGK1L The nucleotides 199 to 339 were removed from thet-PA cDNA by site-directed mutagenesis. A plasmid with the recombinantDNA having the domain composition GK1L was isolated by colonyhybridization with mutagenesis primer 2 and sequenced. This was carriedout as follows: The filters with immobilized DNA were pre-hybridized for4 hours at 65° C. in 0.2% SDS, 1.0% Sarkosyl®, 4x SET (0.6 mmol/1 NaCl,0.2 mol/l Tris-HCl, pH 8.0, 4 mmol/l EDTA) and 4x Denhardt's solution(0.08% Ficoll®, 0.08% polyvinylpyrrolidone, 0.08% bovine serum albumin).

The hybridization was carried out for 12 hours at 46° C. in 0.2% SDS,1.0% Sarkosyl®, 4x SET, 4x Denhardt's and with 5×106 cpm kinasedmutagenesis primer per filter. The filters were washed 3×5 minutes atroom temperature, afterwards they were washed 1×10 minutes at 37° C. and1×5 minutes at 50° C. in 4x SET, 0.2% SDS.

EXAMPLE 2 Immunological characterization of GK1L from CHO cells

t-PA cDNA and GKIL cDNA were inserted into the single BamHI cleavagesite of the plasmid pKCR (Proc. Natl. Acad. Sci. USA 78 (1981),1527-1531) as XbaI-HindIII fragments from which the plasmidspKCR-FGK1K2L and pKCR-GK1L were formed. For this the ends of thefragments were filled up with the Klenow fragment of polymerase I. BothcDNAs contained seven authentic nucleotides at the 5' end and their ownpolyadenylation sites were missing. Both plasmids imparted bacteria withresistance against the antibiotic ampicillin (Amp). The expression ofboth cDNAs is driven by the SV40 early promoter. On the cDNA this isfollowed in the plasmid by the large intron of rabbit-β-globin andpolyadenylation sites of rabbit-β-globin and SV40. In order to isolatethis expression cassette pKCR-FGK1K22L and pKCR-GK1L were linerarized bypartial cleavage with SalI and then cut with AatII and the protrudingends were degraded with nuclease S1. This fragment was isolated from alow-melting agarose gel and ligated into the filled-up single EcoRIcleavage site of pAdD26SV(A) (J. Mol. Biol. 159 (1982) 601-621).pAdD26SV(A) contains an expression cassette for mouse cDNA which isdriven by the major late promoter of adenovirus 2 (AMLP), the SV40origin of replication and it imparts bacteria with resistance againstthe antibiotic tetracyclin. The orientation of the expression cassettefor t-PA in the resulting plasmids pSV-FGK1K2L and pSV-GK1L was examinedby restriction analysis. FIG. I shows diagrammatically the production ofthe plasmid pSV-GKIL as well as the position of individual elements onthe plasmid.

CHO dhfr⁻ cells (ECACC 88072103) were transformed with the recombinantvectors pSV-FGK1K2L and pSV-GK1L (Proc. Natl. Acad. Sci. USA 76 (1979),4350-4354). For this calcium phosphate precipitates were prepared with20 μg pSV-FGK1K2L or pSV-GK1L in a volume of 4 ml (Virology 52 (1973),456-467). 1 ml of the precipitate was added to 3× 10⁵ to 1×10⁶ cells in10 ml medium. The cells were incubated for 8-16 hours, the medium wasthen removed, the cells washed with 10 ml TBS (25 mmol/l Tris-HCl, pH7.4, 137 mmol/l NaCl, 5 mmol/l KCl, 0.6 mmol/l NaH₂ PO₄) and thenincubated in a suitable medium. 48 hours after transfection the CHOdhfr⁻ cells (ECACC 88072103) were diluted 1:10 and then cultured in aselection medium (J. Mol. Biol. 159 (1982), 601-621). The clones whichformed were trypsinized 2 to 3 weeks after the transfection with the aidof a cloning cylinder, they were grown to a mass culture and thesupernatants were examined by ELISA (Gene 51 (1987) 31-41) for t-PAimmunoreactivity. Positive clones were incubated in a medium containing20 nmol/l methotrexate. Methotrexate-resistant colonies appeared after 2weeks. They were cultured to confluence and exposed to 100 nmol/lmethotrexate in the medium. Resistant cells were exposed to methotrexateconcentrations which were increased stepwise (300 nmol/l, 500 nmol/l, 1μmol/l and 5 μmol/l). Clones were isolated by limit dilution and thosewhich produced t-PA best were selected.

CHO cells which showed constitutive secretion of wild-type t-PA or GK1Lwere cultured in DMEM medium (Dulbecco's modified Eagle Medium)supplemented with 10% fetal calf serum in the presence and absence ofaprotinin (50 μg/ml). The supernatants were adjusted with HCl to 0.3mol/l arginine pH 7.5 and applied to an ETI sepharose column (J. Biol.Chem. 259 (1984) 11635-11638). The proteins were eluted with 20 mmol/lcitrate buffer, pH 2.5 and subsequently dialysed against 20 mmol/l TrisHCl, pH 7.5.

Aliquots of the purified proteins to which 10 μg cytochrome C was addedwere precipitated for one hour with 4 volumes acetone at 20° C. andsubsequently dissolved in Laemmli sample buffer. The protein sampleswere boiled for 3 minutes and separated on a 12.5% SDS polyacrylamidegel using a discontinuous buffer system (Laemmli, Nature 227 (1970),680-685). After the electrophoresis the gels were electroblotted ontonitrocellulose filters. The filters were washed with TBS and thensaturated at room temperature for 30 to 60 minutes with TBS +0.05% Tween+3% gelatin and finally briefly washed with water. Afterwards themembrane filters were treated for 1 hour at room temperature with a1:1000 dilution of a peroxidase-conjugated goat antibody against humant-PA in TBS +0.5% bovine serum albumin. In order to visualize theimmunocomplexes the filters, after a further three washing steps withTBS, were incubated with a 1:1 solution of 2.5 mmol/ltetramethylbenzidine and 4.5 mmol/l sodium dioctylsulfosuccinate inmethanol and 0.005% hydrogen peroxide in 0.1 mol/l citric acid buffer,pH 5. The Rainbow-Mix (Amersham) which contains the following proteins:myosin, 200 kd, phosphorylase 92.5 kd, bovine serum albumin, 69 kd,ovalbumin, 46 kd, carboanhydrase, 30 kd, trypsin inhibitor, 21.5 kd andlysozyme, 14 kd, was used as a marker for the polyacrylamide gelelectrophoresis.

The culture supernatant of cells which contain the plasmid pSV-GK1Lwhich expresses GK1L gives an immunoreactive band of about 50000 daltons(corresponding to the single-chain form of GK1L), a band of about 31000daltons (corresponding to the L chain) and a band of about 19000 daltons(corresponding to the H chain) when treated with antibodies againstt-PA. When the cell medium contains the protease inhibitor aprotinin theproportion of double-chain molecules decreases in comparison to thesingle-chain molecules.

In contrast t-PA has a band of 65000 to 68000 daltons (corresponding tothe single-chain form) and 2 bands of 34000 or 31000 daltons(corresponding to the H or L chain).

EXAMPLE 3 Comparison of the fibrinogen-stimulated catalytic activitiesof t-PA and GK1L

t-PA and GK1L were concentrated from the supernatants of CHO cells asdescribed in Example 2. In this process the CHO cells which secrete t-PAor GK1L were cultured in the presence or absence of aprotinin (50μg/ml). In order to carry out the activity test the supernatants werediluted 1:250 which resulted in a negligibly small inhibitorconcentration. The plasminogen-cleaving activity was determined by anindirect spectrophotometric test (Thromb. Haemostasis 48 (1982),266-269). t-PA converts plasminogen into the active serine proteaseplasmin which hydrolyses a bond of a chromogenic substrate whoseabsorbance was measured at 405 nm for a period of up to 3 hours. Inmodified experiments tosylated Gly-Pro-Lys-p-nitroanilide (Chromozy® PL)was used as the chromogenic substrate. The tests were carried out at 25°C. in 0.1 mol/l TrisHCl, pH 7.5, 0.15 mol/l Tween 80 and 0.13 μmol/lplasminogen and 0.30 mmol/l Chromozy® PL in the absence or presence offibrinogen cleaved with CNBr (120 μg/ml). The absorbance was determinedat 405 nm as a measure for the release of p-nitrophenol from thechromogenic substrate and recorded as a function of the incubationperiod. The results are shown in FIG. 2.

The plasminogen-cleaving activity of t-PA (isolated from supernatantsfree of aprotinin) is represented by filled-in circles, the activity oft-PA (isolated from supernatants containing aprotinin) is represented byopen circles, the activity of GKIL (isolated from supernatantscontaining aprotinin) is represented by filled-in squares and theactivity of GK1L (isolated from supernatants without aprotinin) isrepresented by open squares. ±CNBr shows the presence or absence offibrinogen fragments treated with CNBr in the test. (+) or (-) showswhether the protein was purified from tissue culture supernatantscontaining aprotinin or free of aprotinin.

From FIG. 2 it can be seen that a stimulation of the catalytic activityof GKIL results in the presence of fibrinogen.

In this case it is surprising that the catalytic activity, with andwithout fibrinogen, of a supernatant of cells which express GK1L issignificantly higher than that of a supernatant of cells expressingwild-type t-PA. If aprotinin is present in the medium the activity ofGK1L is higher in the presence of fibrinogen and lower withoutfibrinogen than without aprotinin.

I claim:
 1. Human tissue type plasminogen activator derivative having anamino acid sequence corresponding to amino acids 50-175 and 262-527 ofFIG.
 3. 2. Human tissue type plasminogen activator derivative of claim1, wherein said derivative is glycosylated at its natural glycosylationsites.
 3. Fibrinolytic agent comprising a human tissue type plasminogenactivator of claim 1 and a carrier.